Trichothec-9-ene-3,4,15-triol,12,13-epoxy-3-chloroacetate useful as an anti-tumor agent

ABSTRACT

Novel mono-, di- and triacylated derivatives of scirpentriol are provided for use as antitumor agents. Also provided are methods for the production of such derivatives.

This application is a division of our co-pending application Ser. No.339,581 filed Jan. 15, 1982, now U.S. Pat. No. 4,413,134, which is adivision of application Ser. No. 240,340 filed Mar. 4, 1981 now U.S.Pat. No. 4,332,732 which is a division of application Ser. No. 137,336filed Apr. 4, 1980 now U.S. Pat. No. 4,284,568.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel trichothecene derivatives, toprocesses for their production and to their use as antitumor agents forthe inhibition of malignant tumors in mammals.

2. Description of the Prior Art

The trichothecene derivatives of the present invention in generalcontain a 9,10 double bond and a 12,13-epoxy function. The basicskeleton and numbering system for this class of trichothecenes is shownbelow. ##STR1## Various examples of both naturally occurring andsemi-synthetic compounds of this class have been described in theliterature. Illustrative of the more relevant publications are thefollowing:

1. The compound anguidine (also called diacetoxyscirpenol) having theformula ##STR2## is disclosed as an antitumor agent in U.K. Pat. No.1,063,255. Phase I clinical trials of anguidine in the United Stateshave been reported in Proc. Amer. Assoc. Cancer Res. 17: 90 (1976) andProc. Amer. Assoc. Cancer Res. 18: 296 (1977). Also disclosed (at leastgenerically) are various derivatives of anguidine such as anguidol (alsocalled scirpentriol or 3α,4β,15-trihydroxy-12,13-epoxytrichothec-9-ene),monodesacetylanguidine (presumably15-acetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene ormonoacetoxyscirpendiol) and esters of anguidine, anguidol andmonodesacetylanguidine.

Monoacetoxyscirpenol and certain esters of scirpentriol (not includingany of the presently claimed compounds) are also disclosed in J. Agric.Food Chem. 24(1): 97-103 (1976) as mycotoxins.

2. Japanese Published Applications No. J4 9,134,891 and No. J4 9,134,892disclose T2 and HT2 toxins of the formula ##STR3## wherein R is --OH or##STR4## The compounds are said to be useful as antiviral agents.

3. U.S. Pat. No. 4,129,577 discloses anguidine derivatives of theformula ##STR5## wherein R₁ is H or ##STR6## and R is an alkyl oraromatic group or is an acyl group ##STR7## in which R¹ is an aliphatic,cycloaliphatic or aromatic group or a carbamate group --CONH--R¹. Thecompounds are useful as cytotoxic agents.

4. U.S. Pat. No. 3,428,652 discloses anguidine derivatives of theformula ##STR8## wherein R₁ is H and R₂ is methyl or, R₁ and R₂ togetherrepresent propylene, and Hal is Cl, Br or I. The compounds are reportedto have antitumor activity.

5. The 12,13-epoxytrichothecenes of the general formula ##STR9## whereinR¹ and R³ are H, OH or esterified OH and R² is OH, ═O or esterified OHare described in Biochemical and Biophysical Research Communications57(3): 838-844 (1974) as inhibitors of protein synthesis. None of theester derivatives of the present invention are disclosed in thispublication.

SUMMARY OF THE INVENTION

The present invention provides novel mono-, di- and triacylatedderivatives of scirpentriol which may be represented by the generalformula ##STR10## in which R₁, R₂ and R₃ are hydrogen or the residue ofcertain ester groups and at least one of R₁, R₂ or R₃ is other thanhydrogen.

More specifically, the present invention provides (1) monoacylated esterderivatives of the formula ##STR11## in which R₃ is --COCH₂ Cl,--COCH═CHCH₃, --COCCH₃ ═CH₂, --COC₆ H₅ or --COCHClCH₃ ; ##STR12## inwhich R₁ is --COCH₂ Cl; and ##STR13## in which R₂ is --COCH₂ Cl; (2)diacylated ester derivatives of the formulae ##STR14## in which R₂ andR₃ are each --COCH₂ Cl; R₂ and R₃ are each --COCH═CHCH₃ ; R₂ is --COCH₂Cl and R₃ is --COCH₃ ; or R₂ is --COCH₂ Cl and R₃ is --COCCH₃ ═CH₂ ; and##STR15## in which R₁ and R₃ are each --COCH₂ Cl; (3) the triacylatedester derivative of the formula ##STR16## in which R₁, R₂ and R₃ areeach --COCH₂ Cl; and (4) the monoacylated ester epoxy derivative of theformula ##STR17## in which R₃ is --COCH═CHCH₃.

The ester derivatives of formula II-V are antitumor agents for treatmentof malignant tumors in mammals.

As denoted by the structural formulae above, the compounds of formulaeII-V all have the α-configuration at the 3-substituent and theβ-configuration at the 4-substituent.

DETAILED DESCRIPTION

The ester derivatives of the present invention may be prepared bymethods known in the art. Illustrative of such methods are the reactionschemes shown below. ##STR18##

EXPLANATION OF SCHEMES 1-7

In general, the esters of the present invention may be prepared byacylation of scirpentriol or the 3-tetrahydropyran-protected derivatives2 and 3. To prepare 3-tetrahydropyran (THP)-protected derivatives 2 and3, the 3α-OH group of anguidine is protected as by conversion to atetrahydropyranyl ether 1 and this 3α-OTHP derivative is then subjectedto partial basic hydrolysis to give a mixture of the 4β-OH (3) and4β,15-OH (2) derivatives.

In Scheme 2, scirpentriol may be acylated in accordance withconventional methods with a suitable acylating derivative of acarboxylic acid R--COOH to produce a mixture of the various mono-, di-and triacylated derivatives. The desired product is then separated fromthe product mixture as by silica gel column chromatography. Theacylation is typically carried out with an acid halide or acidanhydride, preferably in the presence of an organic base such aspyridine or lutidine. An inert organic solvent such as methylenechloride may be employed or the organic base may also serve as areaction solvent.

Scheme 3 shows the acylation of diol intermediate 2 with two or moreequivalents of acylating agent followed by hydrolysis of the 3α-OTHPgroup to give 4,15-diacylated esters of formula IIIA having R₂ =R₃. Theacylation procedure is carried out by conventional procedures such asdescribed for Scheme 2.

Scheme 4 illustrates a procedure for preparing a 4,15-diacylated esterof formula IIIA where R₃ is --COCH₃. In this procedure starting material5 is acylated as described above to give a 3α-THP derivative which ishydrolyzed to produce the desired product.

If diol intermediate 2 is acylated with less than two equivalents ofacylating agent as in Scheme 5, there may be produced after the usualde-blocking step a mixture of 4- and 15-monoacylated products. Theseproducts can then be separated as by chromatography.

Scheme 6 shows that the monoacylated 3α-THP intermediate as produced inScheme 5 can be treated with a second acylating agent to give after thede-protection step a diacylated ester of formula IIIA where R₂ ≠R₃.

Finally, Scheme 7 illustrates epoxidation of a 15-monoacylated esterwith metachloroperbenzoic acid to give the corresponding 9,10-epoxide.

BIOLOGICAL ACTIVITY

Representative compounds of the present invention were tested forantitumor activity against the transplantable mouse tumors P-388leukemia, L-1210 leukemia and Lewis lung carcinoma and the results ofthese tests are shown below in Tables I-XVIII. The methodology usedgenerally followed the protocols of the National Cancer Institute (see,for example, Cancer Chemotherapy Rep. Part 3, 3: 1-103 (1972)). Theessential experimental details are given at the bottom of the tables.

                  TABLE 1                                                         ______________________________________                                        Effect of Compound of Example 1E on P-388 Leukemia                                                      Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Compound mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270*                                                                             0.4       15.5   172   +0.5    6/6                                            0.2       12.5   139   +0.8    6/6                                   NSC-141537                                                                             0.8       12.5   139   +0.3    6/6                                   (Anguidine)                                                                            0.4       11.0   122   -0.3    6/6                                            0.2       9.5    100   -0.3    6/6                                            0.1       9.0    100   -0.3    6/6                                   Compound of                                                                            6.4       18.5   206   -0.3    6/6                                   Example 1E                                                                             3.2       16.0   178   -0.3    6/6                                            1.6       14.0   156   -3.1    6/6                                            0.8       12.5   139   +1.5    6/6                                            0.4       12.5   139   +0.4    6/6                                            0.2       10.5   117    0.4    6/6                                   Control  Saline    9.0    --    +0.7    10/10                                 ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host CDF.sub.1 ♂ mice.                                                   Treatment QD 1→9.                                                      Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control × 100.                           Criteria T/C≧125 considered significant antitumor effect.              *NSC38270 used in this and the following tables is a crude (˜40%)       preparation of olivomycin A which is used as a reference in screening of      anguidine derivatives.                                                   

                  TABLE II                                                        ______________________________________                                        Effect of Compound of Example 1C on P-388 Leukemia                                                      Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       14.0   156   +0.6    6/6                                            0.2       12.5   139   +0.8    6/6                                   NSC-141537                                                                             1.6       16.5   183   -0.1    6/6                                   (Anguidine)                                                                            0.8       15.0   167   +0.4    6/6                                            0.4       13.0   144   +1.3    6/6                                            0.2       11.5   128   +0.5    6/6                                            0.1       11.0   122   +1.0    6/6                                            0.05      9.5    106   +0.8    6/6                                   Compound of                                                                            6.4       14.5   161   -0.5    6/6                                   Example 1C                                                                             3.2       14.5   161   +0.3    6/6                                            1.6       12.5   139   +0.4    6/6                                            0.8       12.0   133   +0.6    6/6                                            0.4       11.0   122   +0.3    6/6                                            0.2       10.5   117   +0.3    6/6                                            0.1       10.0   111   +0.6    6/6                                            0.05      10.0   111   +0.8    6/6                                            0.025     9.0    100   +1.0    6/6                                            0.0125    9.0    100   +0.8    6/6                                   Control  0.5       9.0    --    +0.5    10/10                                 ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells imp1anted i.p.                          Host CDF.sub.1  ♀ mice.                                                Treatment QD 1→9.                                                      Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control × 100.                           Criteria T/C≧125 considered significant antitumor effect.         

                  TABLE III                                                       ______________________________________                                        Effect of Compound of Example 9 on P-388 Leukemia                                                        Effect                                                                              Average Survi-                                        Dose       MST    MST   Weight  vors                                 Compound mg/kg/day  Days   % T/C Change, g                                                                             Day 5                                ______________________________________                                        NSC-38270                                                                              0.4        13.0   144   -0.7    6/6                                           0.2        11.0   122   -0.5    6/6                                  NSC-141537                                                                             3.2        17.5   194   +0.6    6/6                                  (Anguidine)                                                                            1.6        17.5   194   +0.2    6/6                                           0.8        15.0   167   +0.4    6/6                                           0.4        14.5   161   +0.5    6/6                                           0.2        13.5   150   +1.0    6/6                                           0.1        11.0   122   +0.1    6/6                                           0.05       11.0   122   +0.2    5/5                                           0.025       9.5   106   +0.5    6/6                                  Compound of                                                                            3.2        16.5   183   +0.8    6/6                                  Example 9                                                                              1.6        16.0   178   +1.0    6/6                                           0.8        16.0   178   +1.3    6/6                                           0.4        14.5   161   +0.4    6/6                                           0.2        12.0   133   +0.7    6/6                                           0.1        12.0   133   +0.8    6/6                                           0.05       10.5   117   +0.3    6/6                                           0.025      10.5   117   +0.3    6/6                                           0.0125     10.0   111   +0.3    6/6                                           0.00625    10.0   111   +0.8    6/6                                  Control  DMSO-HPC    9.0   --    +0.4    10/10                                ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host CDF.sub.1 ♀ mice.                                                 Treatment QD 1→9.                                                      Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control = 100.                                 Criteria T/C ≧125 considered significant antitumor effect.        

                  TABLE IV                                                        ______________________________________                                        Effect of Derivatives on P-388 Leukemia                                                                 Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Compound mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       12.5   139   +0.3    6/6                                            0.2       11.0   122   +1.3    6/6                                   NSC-141537                                                                             1.6       17.0   189   +0.8    6/6                                   (anguidine)                                                                            0.8       15.0   167   +1.0    6/6                                            0.4       13.5   150   +1.6    6/6                                            0.2       13.0   144   +1.5    6/6                                            0.1       11.0   122   +1.4    6/6                                            0.05      11.0   122   +1.8    6/6                                   Compound of                                                                            6.4       14.0   156   +0.9    6/6                                   Example 1D                                                                             3.2       13.0   144   +1.3    6/6                                            1.6       12.5   139   +2.8    6/6                                            0.8       12.5   139   +2.3    6/6                                            0.4       11.5   128   +1.6    6/6                                            0.2       10.5   117   +1.2    6/6                                   Compound of                                                                            6.4       17.5   194   +1.2    6/6                                   Example 1B                                                                             3.2       14.0   156   +3.0    5/5                                            1.6       13.0   144   +1.5    6/6                                            0.8       12.0   133   +1.7    6/6                                            0.4       12.0   133   +1.7    6/6                                            0.2       10.0   111   +1.4    6/6                                   Compound of                                                                            6.4       16.0   178   -0.9    6/6                                   Example 8                                                                              3.2       14.0   156   -0.6    6/6                                            1.6       13.0   144   -0.1    6/6                                            0.8       12.0   133   -0.3    6/6                                            0.4       12.5   139   -0.7    6/6                                            0.2       11.5   128   -0.3    6/6                                            0.1       13.0   144   -0.6    6/6                                            0.5       11.0   122   -0.5    6/6                                            0.025     10.0   111   -0.1    6/6                                            0.0125    10.0   111   -0.4    6/6                                   Control  Saline           --    +0.5    10/10                                 ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host CDF.sub.1 ♂ mice.                                                   Treatment QD 1→9.                                                      Evaluation MST = median survival time.                                        Effect % T/C = MST Treated/MST control × 100.                           Criteria T/C≧125 considered significant antitumor effect.         

                  TABLE V                                                         ______________________________________                                        Effect of Compound of Example 1A on P-388 Leukemia                                                      Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Compound mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       13.0   144   +0.1    5/5                                            0.2       11.0   122   +0.3    6/6                                   NSC-141537                                                                             3.2       17.5   194   +0.7    6/6                                   (anguidine)                                                                            1.6       17.0   189   +0.7    6/6                                            0.8       14.0   156   +1.8    6/6                                            0.4       13.5   150   +1.9    6/6                                            0.2       12.0   133   +0.8    6/6                                            0.1       11.0   122   +1.0    6/6                                            0.05      11.5   128   +1.2    6/6                                            0.025     10.0   111   +1.3    6/6                                   Compound of                                                                            12.8      Tox    Tox   Tox     2/6                                   Example 1A                                                                             6.4       Tox    Tox   Tox     2/6                                            3.2       17.5   194   +0.5    6/6                                            1.6       17.0   189   +1.3    6/6                                            0.8       14.5   161   +1.8    6/6                                            0.4       16.0   178   +1.4    6/6                                            0.2       14.0   156   +0.8    6/6                                            0.1       13.0   144   +1.3    6/6                                            0.05      13.5   150   +1.3    6/6                                            0.025     12.5   139   +1.4    6/6                                            0.0125    10.5   117   +1.3    6/6                                            0.00625   10.5   117   + 1.7   6/6                                   Control  Saline     9.0   --    +0.6    10/10                                 ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host CDF.sub.1 mice.                                                          Treatment Daily, QD 1→9.                                               Tox Toxicity <4/6 survivors, Day 5.                                           Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control × 100.                           Criteria T/C ≧125 considered significant antitumor effect.        

                  TABLE VI                                                        ______________________________________                                        Effect of Compound of Example 2 on P-388 Leukemia                                                      Effect                                                                              Average                                                Dose      MST    MST   Weight  Survivors                              Material                                                                              mg/kg/day Days   % T/C Change, g                                                                             Day 5                                  ______________________________________                                        NSC-38270                                                                             0.4       13.0   153   +0.4    6/6                                    A649    0.2       10.0   118   +1.2    6/6                                    NSC-141537                                                                            1.6       16.0   188   +1.0    6/6                                    Anguidine                                                                             0.8       14.0   165   +0.9    6/6                                            0.4       13.0   153   +1.3    6/6                                            0.2       12.0   141   +1.0    6/6                                            0.1       11.0   129   +0.4    6/6                                            0.05      10.0   118   +1.1    6/6                                    Compound                                                                              6.4       18.0   212   -0.3    5/6                                    of Exam-                                                                              3.2       16.5   194   +1.0    6/6                                    ple 2   1.6       15.5   182   +1.1    6/6                                            0.8       14.0   165   +1.3    6/6                                            0.4       13.0   153   +0.8    6/6                                            0.2       13.0   153   +0.2    6/6                                            0.1       11.5   135   +0.8    6/6                                            0.05      12.5   147   +0.9    6/6                                            0.025     10.0   118   +1.1    6/6                                            0.0125    9.0    106   +2.4    6/6                                            0.00625   9.0    106   +2.3    6/6                                            0.003125  9.0    106   +3.1    6/6                                    Control Saline    8.5    --    +3.1    10/10                                  ______________________________________                                          Tumor inoculum 10.sup.6 ascites cells implanted i. p.                        Host CDF.sub.1 ♂ mice.                                                   Treatment QD 1→9.                                                      Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control × 100.                           Criteria T/C≧125 considered significant antitumor effect.         

                                      TABLE VII                                   __________________________________________________________________________    Effect of Compound of Example 2 on P-388 Leukemia                                                    Effect                                                                              Average                                                       Dose, IP                                                                             MST                                                                              MST   Weight                                                                              Survivors                                  Material                                                                             Treatment                                                                           mg/kg/day                                                                            Days                                                                             % T/C Change, g                                                                           Day 5 (30)                                 __________________________________________________________________________    NSC 38270                                                                            Days 1→9                                                                     0.4    11.5                                                                             128   -1.5  6/6                                                     0.2    10.0                                                                             111   -0.9  6/6                                        Anguidine                                                                            Day 1 only                                                                          20     Tox                                                                              Tox   Tox   0/6                                        NSC 141537   16     Tox                                                                              Tox   Tox   0/6                                                     12     Tox                                                                              Tox   Tox   2/6                                                     8      Tox                                                                              Tox   Tox   3/6                                               Days 1→5                                                                     5      Tox                                                                              Tox   Tox   3/6                                                     4      15.0                                                                             167   +0.3  6/6                                                     3      14.0                                                                             156   +0.3  6/6                                                     2      13.0                                                                             144   +0.2  6/6                                               Days 1→9                                                                     2.4    16.0                                                                             178   +1.3  6/6                                                     1.6    16.0                                                                             178   +0.6  5/5                                        Compound of                                                                          Day 1 only                                                                          60     Tox                                                                              Tox   Tox   0/6                                        Example 2    45     Tox                                                                              Tox   Tox   0/6                                                     30     Tox                                                                              Tox   Tox   1/6                                                     20     Tox                                                                              Tox   Tox   0/6                                               Days 1→5                                                                     12     Tox                                                                              Tox   Tox   2/6                                                     10     Tox                                                                              Tox   Tox   1/6                                                     8      13.0                                                                             144   -1.5  5/6                                                     6.4    13.5                                                                             150   -0.8  4/6                                               Days 1→9                                                                     9.0    Tox                                                                              Tox   Tox   3/6                                                     6.4    12.0                                                                             133   +0.3  6/6                                        Control      Saline  9.0                                                                             --    +2.2  10/10                                      __________________________________________________________________________     Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host CDF.sub.1 ♂ mice.                                                   Tox <4/6 survivors Day 5.                                                     Evaluation MST = median survival time.                                        Effect % T/C = (MST treated/MST control) × 100.                         Criteria % T/C≧125 considered significant antitumor activity.     

                  TABLE VIII                                                      ______________________________________                                        Effect of Compound of Example 2 on P-388 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/inj Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-141537                                                                             1.6       11.0   183   +0.5    6/6                                   (Anguidine)                                                                            0.8       8.5    142   +1.2    6/6                                            0.4       8.5    142   +1.2    6/6                                            0.2       8.0    133   +1.5    6/6                                            0.1       7.0    117   +1.9    6/6                                             0.05     7.0    117   +2.6    6/6                                   Compound of                                                                            12.8      8.5    142   -0.9    4/6                                   Example 2                                                                              6.4       10.5   175   -0.2    6/6                                            3.2       9.5    158   +1.0    6/6                                            1.6       9.5    158   +1.8    6/6                                            0.8       8.5    142   -1.3    6/6                                            0.4       8.5    142   -0.7    6/6                                            0.2       7.0    117   +0.3    6/6                                            0.1       7.0    117   +0.3    6/6                                   Control  Saline    6.0    --    +2.5    10/10                                 ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host BDF.sub.1 ♂ mice.                                                   Treatment Daily, QD 1→9.                                               Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control × 100.                           Criteria T/C≧125 considered significant antitumor effect.         

                  TABLE IX                                                        ______________________________________                                        Effect of Compound of Example 4 on P-388 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       12.0   133   +0.7    6/6                                   (A-649)  0.2       11.0   122   -0.4    6/6                                   NSC-141537                                                                             1.6       17.0   189   +1.3    5/5                                   (anguidine)                                                                            0.8       14.0   156   +1.7    5/6                                            0.4       14.0   156   +1.9    6/6                                            0.2       12.0   133   +1.5    6/6                                            0.1       11.5   128   +0.8    6/6                                            0.05      10.0   111   +0.3    6/6                                   Compound of                                                                            6.4       Tox    Tox   Tox     1/6                                   Example 4                                                                              3.2       20.0   222   -1.9    5/6                                            1.6       17.0   189   -0.9    6/6                                            0.8       14.5   161   +0.9    6/6                                            0.4       13.0   144   +0.2    5/6                                            0.2       12.5   139   0       6/6                                            0.1       13.0   144   0       6/6                                            0.05      11.0   122   -0.8    6/6                                            0.025     11.0   122   -0.3    6/6                                            0.0125    11.0   122   -0.4    6/6                                   Control  Saline     9.0   --    +0.3    10/10                                 ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host CDF.sub.1 ♂  mice.                                                  Treatment Daily, QD 1→9.                                               Tox Toxicity, 4/6 survivors Day 5.                                            Evaluation MST = median survival time.                                        Effect % T/C = MST treated/MST control × 100.                           Criteria T/C ≧125 considered significant antitumor effect.        

                  TABLE X                                                         ______________________________________                                        Effect of Compound of Example 4 on L-1210 Leukemia                                                     Effect                                                                              Average                                                Dose, IP  MST    MST   Weight  Survivors                              Material                                                                              mg/kg/day Days   % T/C Change, g                                                                             Day 5 (30)                             ______________________________________                                        Anguidine                                                                             2.0       11.0   157   -0.8    6/6                                    NSC     1.6       11.0   157   -0.3    6/6  (1/6)                             141537  1.2       11.0   157   -0.3    6/6                                            0.8       11.0   157   -0.3    6/6                                            0.4       10.0   143   -0.1    6/6                                            0.2       9.0    129   +0.5    6/6  (1/6)                             Compound                                                                              1.6       12.0   171   -0.8    4/6  (2/6)                             pf Exam-                                                                              0.8       10.0   143   -0.3    6/6                                    ple 4   0.4       9.5    136   -0.8    6/6                                            0.2       9.0    129   -0.3    6/6                                    Control Saline    7.0    --    +0.9    10/10                                  ______________________________________                                         Tumor inoculum 10.sup.6 ascites cells implanted i.p.                          Host BDF.sub.1 ♂ mice.                                                   Treatment QD 1→9.                                                      Tox <4/6 mice alive on Day 5.                                                 Evaluation MST = median survival time.                                        Effect % T/C = (MST treated/MST control) × 100.                         Criteria % T/C≧125 considered significant antitumor activity.     

                  TABLE XI                                                        ______________________________________                                        Effect of Derivatives on L-1210 Leukemia                                                                Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        Anguidine                                                                              2.0       6.0     86   -0.9    4/6                                   NSC 141537                                                                             1.6       6.0    86    -1.3    6/6                                            1.2       11.0   157   -1.1    5/6                                            0.8       11.0   157   +1.0    5/6                                   Compound of                                                                            4.0       Tox    Tox   Tox     0/6                                   Example 4                                                                              3.2       Tox    Tox   Tox     0/6                                            2.4       Tox    Tox   Tox     2/6                                            1.6       Tox    Tox   Tox     3/6                                   Compound of                                                                            2.4       6.0    86    -1.5    5/6                                   Example 3                                                                              1.6       6.0    86    -1.3    6/6                                            1.2       8.0    114 -0.6                                                                            6/6                                                    0.8       12.0   171   -2.6    6/6                                   Compound of                                                                            0.6       Tox    Tox   Tox     3/6                                   Example 6                                                                              0.4       10.0   143   -1.1    5/6                                            0.3       10.5   150   -1.0    4/6                                            0.2       10.0   143   +0.1    5/6                                   Control  Saline    7.0    --    +2.4    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascitic cells implanted ip                           Host: BDF.sub.1 ♀ mice.                                                Treatment: QD 1→9                                                      Tox: <4/6 survivors Day 5                                                      Evaluation: % T/C = MST treated/MST control × 100.                     Criteria: % T/C ≧125 considered significant antitumor effect.     

                  TABLE XII                                                       ______________________________________                                        Effect of Compound of Example 3 on P-388 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vor                                   Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       12.5   139   -0.6    6/6                                            0.2       11.0   122   +0.3    6/6                                   NSC-141537                                                                             1.6       15.0   167   -0.2    6/6                                   (anguidine)                                                                            0.8       14.0   156   +0.5    6/6                                            0.4       17.0   189   +0.2    6/6                                            0.2       16.5   183   -0.5    6/6                                            0.1       11.0   122   +0.7    6/6                                            0.05      10.5   117   +0.6    6/6                                   Compound of                                                                            6.4        7.0    78   -2.0    5/6                                   Example 3                                                                              3.2        7.5    83   -1.0    6/6                                            1.6       20.0   222   -0.6    6/6                                            0.8       19.5   217   +0.1    6/6                                            0.4       17.0   189   +0.5    /66                                            0.2       15.5   172   -0.1    6/6                                            0.1       14.5   161   +0.1    6/6                                            0.05      13.0   144   +0.1    6/6                                            0.025     12.0   133   -0.2    6/6                                            0.0125    10.5   117   +0.6    6/6                                   Control  Saline     9.0   --    0       10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                         Host: CDF.sub.1 ♀ mice.                                                Treatment: QD 1→9.                                                     Tox: Toxicity, <4/6 survivors, Day 5.                                         Evaluation: MST = median survival time.                                       Effect: % T/C = MST treated/MST control × 100.                          Criteria: T/C ≧125 considered significant antitumor effect.       

                  TABLE XIII                                                      ______________________________________                                        Effect of Compound of Example 5 on P-388 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       11.0   122   -0.4    6/6                                            0.2       9.5    106   +0.1    6/6                                   Compound of                                                                            6.4       15.0   167   -0.4    5/6                                   Example 5                                                                              3.2       14.5   161   0       6/6                                            1.6       13.0   144   +1.5    5/6                                            0.8       10.0   111   +0.8    6/6                                            0.4       9.0    100   +1.2    6/6                                            0.2       9.5    106   +2.8    6/6                                            0.1       10.0   111   +2.9    6/6                                             0.05     9.0    100   +3.1    6/6                                   NSC-141537                                                                             1.6       15.0   167   +1.5    6/6                                   (Anguidine)                                                                            0.8       15.0   167   +1.6    6/6                                            0.4       14.0   156   +1.2    6/6                                            0.2       12.0   133   +0.8    6/6                                            0.1       10.5   117   +1.4    6/6                                             0.05     10.0   111   +1.5    6/6                                   Control  Saline    9.0    --    +3.7    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                         Host: CDF.sub.1 ♀ mice.                                                Treatment: QD 1→9.                                                     Evaluation: MST =  median survival time.                                      Effect: % T/C = MST treated/MST control × 100.                          Criteria: T/C ≧125 considered significant antitumor effect.       

                  TABLE XIV                                                       ______________________________________                                        Effect of Compound of Example 6 on P-388 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC-38270                                                                              0.4       11.0   138   -1.0    6/6                                            0.2       10.0   125   -0.2    6/6                                   Anguidine                                                                              1.6       15.0   188   +0.6    6/6                                   NSC 141537                                                                             0.8       13.0   163   +0.7    6/6                                            0.4       13.0   163   +0.3    6/6                                            0.2       12.0   150   +0.8    6/6                                            0.1       10.0   125   +0.1    6/6                                             0.05     10.0   125   +0.4    6/6                                   Compound of                                                                            12.8      Tox    Tox   Tox     0/6                                   Example 6                                                                              6.4       Tox    Tox   Tox     0/6                                            3.2       Tox    Tox   Tox     0/6                                            1.6       Tox    Tox   Tox     0/6                                            0.8        6.0   75    -1.9    4/6                                            0.4       18.0   225   -1.1    6/6                                            0.2       15.5   194   -0.5    6/6                                            0.1       14.0   175   -0.7    6/6                                   Control  Saline     8.0   --    -0.4    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascitic cells implanted i.p.                         Host: CDF.sub.1 ♀ mice.                                                Treatment: Daily, QD 1→9.                                              Tox: <4/6 survivors Day 5.                                                    Evaluation: MST = median survival time.                                       Effect: T/C = (MST treated/MST control) × 100.                          Criteria: % T/C ≧125 considered significant antitumor activity.   

                  TABLE XV                                                        ______________________________________                                        Effect of Compound of Example 10 on P-388 Leukemia                                                      Effect                                                                              Average Survi-                                         Dose      MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5                                 ______________________________________                                        NSC 38270                                                                              0.4       10.5   117   -1.2    6/6                                            0.2       10.5   117   -0.8    6/6                                   Anguidine                                                                              1.6       17.5   195   -0.7    6/6                                   NSC 141537                                                                             0.8       15.0   167   +0.7    6/6                                            0.4       14.0   156   +0.2    6/6                                            0.2       12.0   133   -0.3    6/6                                            0.1       10.5   117   +0.8    6/6                                             0.05     10.5   117   +0.4    6/6                                   Compound of                                                                            12.8      16.5   183   -0.8    6/6                                   Example 10                                                                             6.4       15.0   167   +0.3    6/6                                            3.2       16.0   178   +0.8    6/6                                            1.6       12.0   133   -0.1    6/6                                            0.8       12.0   133   +0.2    6/6                                            0.4       11.0   122   +0.7    6/6                                   Control  Saline     9.0   --    -1.8    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascitic cells implanted ip                           Host: CDF.sub.1 ♂ mice.                                                  Treatment: QD 1→9.                                                     Tox: <4/6 survivors Day 5                                                     Evaluation: MST = median survival time                                        Effect: % T/C = (MST treated/MST control) × 100.                        Criteria: % T/C ≧125 considered significant antitumor activity    

                  TABLE XVI                                                       ______________________________________                                        Effect of Compound of Example 7 on P-388 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose, IP  MST    MST   Weight  vors                                  Material mg/kg/inj Days   % T/C Change, g                                                                             Day 5 (3)                             ______________________________________                                        NSC 38270                                                                              0.4       9.0      100 +0.3    6/6                                            0.2       9.0      100 +2.8    6/6                                   Anguidine                                                                              1.6       14.5     161 +1.4    6/6                                   NSC 141537                                                                             0.8       13.0     144 +1.7    6/6                                            0.4       12.0     133 +1.8    6/6                                            0.2       10.0     111 +1.4    6/6                                            0.1       9.0      100 +2.3    6/6                                            0.05      9.0      100 +2.6    6/6                                   Compound of                                                                            12.8      Tox      Tox Tox     0/6                                   Example 7                                                                              6.4       Tox      Tox Tox     0/6                                            3.2       Tox      Tox Tox     0/6                                            1.6       18.0     200 -1.3    4/6                                            0.8       17.5     194 -0.5    6/6                                            0.4       15.0     167 -0.1    6/6                                            0.2       12.0     133 +0.3    6/6                                            0.1       12.0     133 +0.3    6/6                                            0.05      11.0     122 +1.2    6/6                                            0.025     10.0     111 +1.1    5/6                                   Control  Saline    9.0      -   +4.0    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascites cells implanted i.p.                          Host: CDF.sub.1 ♀ mice.                                               Treatment: QD 1→9                                                      Evaluation: MST = median survival time.                                       Effect: % T/C = (MST treated/MST control) × 100.                        Criteria: % T/C ≧125 considered significant antitumor effect.     

                  TABLE XVII                                                      ______________________________________                                        Effect of Compound of Example 7 on L1210 Leukemia                                                       Effect                                                                              Average Survi-                                         Dose, IP  MST    MST   Weight  vors                                  Material mg/kg/day Days   % T/C Change, g                                                                             Day 5 (3)                             ______________________________________                                        Anguidine                                                                              2.4       12.0   200   +1.0    6/6                                   NSC 141537                                                                             2.0       11.0   183   +1.9    6/6                                            1.6       10.0   167   +1.4    6/6                                            1.2       10.0   167   +0.9    6/6                                            0.8       10.0   167   +0.9    6/6                                            0.4       9.0    150   +0.1    6/6                                            0.2       8.0    133   +1.3    6/6                                            0.1       8.0    133   +0.8    6/6                                   Compound of                                                                            2.4       TOX    TOX   TOX     1/6                                   Example 7                                                                              2.0       7.0    117   -1.2    3/6                                            1.6       10.0   167   -1.7    5/6                                            1.2       9.5    158   -1.1    6/6                                            0.8       10.0   167   -0.5    6/6                                            0.4       10.0   167   -0.8    6/6                                            0.2       9.0    150   -0.5    5/6                                            0.1       8.0    133   +0.5    6/6                                   Control  Saline    6.0    -     +2.6    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 ascites cells implanted, ip.                         Host: BDF.sub.1 ♀ mice.                                                Treatment: QD 1→9.                                                     Evaluation: MST = median survival time.                                       Effect: % T/C = (MST treated/MST control). × 100                        Criteria: % T/C ≧125 considered significant antitumor activity.   

                  TABLE XVIII                                                     ______________________________________                                        Effect of Compound of Example 7 on Lewis Lung Carcinoma                                                 Effect                                                                              Average Survi-                                         Dose, IP  MST    MST   Weight  vors Day                              Material mg/kg/day Days   % T/C Change  5 (60)                                ______________________________________                                        Anguidine                                                                              1.6       21.0   124   +2.2    10/10                                 NSC 141537                                                                             0.8       21.0   124   +1.8    10/10                                          0.4       23.0   135   +1.4    10/10                                 Compound of                                                                            2.0       21.5   126   -0.5     6/10                                 Example 7                                                                              1.5       21.5   126   -0.3    10/10                                          1.0       22.0   129   +0.5    10/10                                          0.5       22.0   129   +1.2    10/10                                 Control  Saline    17.0   --    -0.6    10/10                                 ______________________________________                                         Tumor inoculum: 10.sup.6 tumor brei cells, ip.                                Host; BDF.sub.1 ♂ mice.                                                  Treatment: QD 1→9.                                                     Tox: <6/10 mice alive on Day 5.                                               Evaluation: MST = median survival time.                                       Effect: % T/C = (MST treated/MST control) × 100.                        Criteria: % T/C ≧125 considered significant antitumor activity.   

EXPLANATION

Each of the 14 derivatives of the present invention was evaluated inmice against P388 leukemia (ip) in parallel with anguidine itself, usinga qd 1→9 dosing schedule (ip). The compounds were all found to be activeand comparable to anguidine with respect to this tumor system.

The compound of Example 2 was evaluated twice against P388 leukemia(Tables VI and VII). As can be seen, the compound for some as yetunexplained reason appeared significantly more active in one test thanthe other.

Five compounds were evaluated in mice against L1210 leukemia (ip). Allof them were active with maximum T/C values of between 150% and 175%using a qd 1→9 dosing schedule.

The compound of Example 7 was also evaluated in mice against Lewis lungcarcinoma (ip). It produced a maximum T/C of 129% when given qd 1→9.

The following examples are not limiting but are intended to beillustrative of this invention. SKELLYSOLVE B is a commerciallyavailable petroleum solvent (Skelly Oil Co.) comprising isomeric hexanesand having a boiling point of 60°-68° C. The main component ofSKELLYSOLVE B is n-hexane. Unless otherwise indicated, all meltingpoints below are uncorrected, all temperatures are in degrees Celsiusand all solvent percentages are by volume. The silica gel used in theexamples (unless otherwise indicated) is SILICAR CC-7 (trademark ofMallinckrodt Chemical Works).

PREPARATION OF STARTING MATERIALS PREPARATION 14β,15-Diacetoxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

A mixture of 4β,15-diacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene(12.81 g, 35 mmol), 2,3-dihydro-4H-pyran (17.5 ml, 189 mmol), andp-toluenesulfonic acid (70 mg, 0.35 mmol) in 150 ml of CH₂ Cl₂ wasstirred at room temperature for 2 h. After addition of 2.1 g of K₂ CO₃,the reaction mixture was diluted with 400 ml of CH₂ Cl₂ and washed withsaturated NaHCO₃ solution and brine. Drying over K₂ CO₃ and removal ofthe solvent gave a colorless oil which crystallized slowly frompetroleum ether to give 11.30 g (72%) of solid. m.p. 93°-94° C.;IR(KBr): 2976, 1746, 1249, 1080, 1040, 988 cm⁻¹.

Anal. Calc'd for C₂₄ H₃₄ O₈ : C, 63.98; H, 7.61. Found: C, 64.35; H,7.58.

PREPARATION 2 3α,4β,15-Trihydroxy-12,13-epoxytrichothec-9-ene

4β,15-Diacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene (15 g) wasstirred for 20 minutes in 300 ml of methanol with 900 ml of 0.3N sodiumhydroxide (Sigg et al. Helv. Chim. Acta, 48, 962-988 (1965). Thesolution was placed on a column containing 1 kg of DOWEX 50 (H⁺ cycle)prepared with 20% methanol in water. The column was eluted with 3 l ofthe same solvent, the eluate concentrated, and the residual aqueoussolution freeze-dried. The powder was dissolved in methanol, mixed with10 g of silica gel, and dried in vacuo. The dry silica gel mixture wasplaced on a column of fresh silica gel (2.5×100 cm) and eluted withmethylene chloride with increasing amounts of methanol. Fractionsappearing homogeneous on TLC plates were dried and crystallized fromethyl acetate. Yield: 7.3 g, m.p. 194°-195° C. IR(KBr): 3490, 3450,3390, 2990-2900 (four peaks), 1675, 960 and 950 cm.sup. -1. [α]_(D) ²²=-15.4° (c=1, acetone).

Anal. Calc'd for C₁₅ H₂₂ O₆ : C, 63.81; H, 7.86. Found: C, 63.71; H,7.80.

Alternatively, the 3-O-tetrahydropyranyl derivative (Preparation 3below) (1 g) was stirred for four hours in 115 ml of 95% ethanol and 23ml of 1N HCl. The solution was azeotropically distilled with theaddition of absolute ethanol, the concentrated ethanolic solutiondiluted with diethyl ether, and the resulting title product separatedfrom ethyl acetate as a gum.

PREPARATION 34β,15-Dihydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

To a solution of4β,15-diacetoxy-3-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene(1.067 g, 2.37 mmol) in 40 ml of tetrahydrofuran and methanol (5:3 v/v)was added 20 ml of 0.3N NaOH solution. After 2.5 h of stirring at roomtemperature, an additional 20 ml of 0.3N NaOH solution was introduced,and stirring was continued for 18.5 h. The resulting solution wasdiluted with CH₂ Cl₂ (200 ml) and washed with water. The aqueous layerwas reextracted with CH₂ Cl₂ (2×50 ml). The combined CH₂ Cl₂ layers werewashed with brine and dried over K₂ CO₃. Removal of the solvent gave 891mg of foam, which was subsequently chromatographed on silica gel.Elution with 1% methanol-CH₂ Cl₂ gave 46 mg (5%) of15-acetoxy-4β-hydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene.A further elution with 5% methanol-CH.sub. 2 Cl₂ gave 808 mg (93%) ofthe title compound as an amorphous solid. IR(KBr): 3457, 2943, 1445,1135, 1125, 1078, 1035, 1020, 978, 957 cm⁻¹.

PREPARATION 415-Acetoxy-4β-hydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

To a solution of4β,15-diacetoxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene(31.14 g, 69.2 mmol) in 800 ml of methanol and tetrahydrofuran (1:1 v/v)was added 400 ml of 1.31N NH₄ OH solution. After 3 days stirring at roomtemperature, 10 ml of concentrated NH₄ OH solution was added to thereaction mixture. Stirring was continued for an additional 4 days. Thevolume of the resulting solution was reduced to 500 ml. Extraction withCH₂ Cl₂, washing with brine, and removal of the solvent gave 37 g of aslightly yellow oil. Chromatography on silica gel (elution with 1%methanol-CH₂ Cl₂) gave 10.65 g (38%) of the title compound as an oil.The NMR and IR spectra of this material were consistent with thestructure of the title compound. IR(KBr): 3430, 2970, 2950, 2875, 1744,1720, 1270, 1248, 1126, 1080, 1040, 972 cm⁻¹.

EXAMPLE 1 A.15-Chloroacetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene

Scirpentriol (3α,4β,15-trihydroxy-12,13-epoxytrichothec-9-ene) (7 g) wasdissolved in 30 ml. of 2,6-lutidine and treated with 10.75 g ofchloroacetic anhydride. After 18 hours the solution was poured on ice.The product of the usual isolation procedure was chromatographed on acolumn of silica gel (2×75 cm) by gradient elution with methylenechloride/methanol. The emerging (Rf 0.1, 0.35, 0.58, 0.80, 0.90)fractions were analyzed on TLC plates appropriately combined, dried andcrystallized from ethyl acetate-ether-hexane. Fractions, Rf 0.35 gave0.640 mg of title product. M.p. 173°. IR(KBr): 3520, 3380, 3800-2900 (4peaks) 1725, 1295, 1060.

Anal. Calc'd for C₁₇ H₂₃ O₆ Cl: C, 56.91; H, 6.46; Cl, 9.88. Found: C,56.85; H, 6.39; Cl, 9.65.

B. 3α-Chloroacetoxy-4β,15-dihydroxy-12,13-epoxytrichothec-9-ene

Fractions Rf 0.58 from Example 1, Part A, gave 480 mg. of title product;m.p. 170°. IR(KBr): 3500, 3000-2900 (five peaks) 1758, 1745, 1210, 1170,1055, 918 cm⁻¹.

Anal. Calc'd for C₁₇ H₂₃ O₆ Cl: C, 56.91; H, 6.46; Cl, 9.88. Found: C,57.03; H, 6.46; Cl, 9.85.

C. 3α,15-Bis(chloroacetoxy)-4β-hydroxy-12,13-epoxytrichothec-9-ene

Fractions Rf 0.8 from Example 1, Part A, gave 1.5 g title product; m.p.161°. IR(KBr): 3480, 3060-2840 (seven peaks) 1765, 1735, 1295, 1200,1165 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₄ O₇ Cl₂ : C, 52.18; H, 5.99; Cl, 16.21. Found:C, 52.41; H, 5.55; Cl, 16.44.

D. 4β-Chloroacetoxy-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene

Fractions with Rf 0.68 from Example 1, Part A, gave 205 mg of titleproduct. The structure of the product was confirmed by PMR spectroscopy.

E. 3α,4β,15-Tri(chloroacetoxy)-12,13-epoxytrichothec-9-ene

Fractions with Rf 0.9 from Example 1, Part A, gave 95 mg. of titleproduct. The structure of the product was confirmed by PMR spectroscopy.

EXAMPLE 24β,15-Bis(chloroacetoxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene A.4β,15-Bis(chloroacetoxy)-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene

A mixture of4β,15-dihydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene(808 mg, 2.21 mmol), chloroacetic anhydride (1.132 g, 6.62 mmol) andpyridine (894 mg, 11.05 mmol) in 100 ml of CH₂ Cl₂ was stirred at roomtemperature for 14 h. The reaction mixture was diluted with 200 ml ofCH₂ Cl₂ and washed with saturated NaHCO₃ solution, 1% HCl solution andbrine. Drying over K₂ CO₃ and removal of the solvent gave 1.058 g (92%)of foam which was homogeneous on TLC. A portion of this material waspurified by silica gel chromatography (elution with 0.5% methanol-CH₂Cl₂) to furnish an analytical sample of title product. IR(KBr): 2955,1762, 1740, 1290, 1186, 1172, 1129, 1080, 1039, 977 cm⁻¹.

B. 4β,15-Bis(chloroacetoxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene

To a solution of4β,15-bis(chloroacetoxy)-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene(858 mg, 1.65 mmol) in 100 ml of 95% ethanol was added 19 ml of 1N HClsolution. The resulting solution was stirred at room temperature for 24hours. The reaction mixture was diluted with CH₂ Cl₂ (300 ml) and washedwith saturated NaHCO₃ solution and brine. Drying over K₂ CO₃ -Na₂ SO₄and removal of the solvent gave 600 mg of foam. Chromatography of thismaterial on silica gel (elution with 1% methanol-CH₂ Cl₂) gave 524 mg(73%) of4β,15-bis(chloroacetoxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene. Ananalytical sample was obtained by recrystallization fromchloroform-diethyl ether, m.p. 139°-141° C. IR(KBr): 3450, 2970, 2913,1758, 1742, 1327, 1293, 1192, 1173, 1083, 1008, 967 cm⁻¹.

Anal. Calc'd. for C₁₉ H₂₄ O₇ Cl₂ : C, 52.42; H, 5.56. Found: C, 52.31;H, 5.34.

Elution of the silica gel column with 2% methanol-CH₂ Cl₂ gave 110 mg(15%) of 15-chloroacetoxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene.

EXAMPLE 315-Acetoxy-4β-chloroacetoxy-3α-hydroxy-12,13-epoxytrichothec-9-ene

A mixture of15-acetoxy-4β-hydroxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene(785 mg, 1.92 mmol), chloroacetic anhydride (492 mg, 2.88 mmol) andpyridine (0.309 ml, 3.84 mmol) in 100 ml of CH₂ Cl₂ was stirred at roomtemperature for 28.5 h. The reaction mixture was diluted with CH₂ Cl₂(200 ml) and washed with saturated NaHCO₃ solution and brine. Dryingover K₂ CO₃ and removal of the solvent gave 931 mg (100%) of a whitefoam. The NMR and IR spectra of this material were consistent with thestructure of15-acetoxy-4β-chloroacetoxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene.

To a solution of15-acetoxy-4β-chloroacetoxy-3α-O-(2'-tetrahydropyranyl)-12,13-epoxytrichothec-9-ene(900 mg, 1.98 mmol) in 100 ml of 95% ethanol was added 19 ml of 1N HClsolution. After 10 h of stirring at room temperature, 200 ml of CH₂ Cl₂was added to the reaction mixture. The aqueous layer which separated wasextracted with 25 ml of CH₂ Cl₂. The combined CH₂ Cl₂ layers were washedwith saturated NaHCO₃ solution and brine. Drying over K₂ CO₃ -Na₂ SO₄and removal of the solvent gave 662 mg of foam. Chromatography on silicagel (elution with 0.5% methanol-CH₂ Cl₂) gave 350 mg (44%) of the titlecompound. Recrystallization from diethyl ether furnished the analyticalsample; m.p. 166°-167.5° C. IR(KBr): 3500, 3040, 3020, 2989, 2918, 1754,1736, 1378, 1330, 1260, 1208, 1167, 1074, 1052, 960 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₅ O₇ Cl: C, 56.83; H, 6.29. Found: C, 57.12; H,6.29.

EXAMPLE 415-(trans-2'-Butenoyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene

A solution containing 366 mg (1 mmol) of3α-O-(2'-tetrahydropyranyl)-4β,15-dihydroxy-12,13-epoxytrichothec-9-eneand 395 mg (5 mmol) of dry pyridine in 50 ml of CH₂ Cl₂ (previouslydried over 4 Å molecular sieves) was cooled at 5° C. To the stirredsolution was added dropwise 261 mg (2.5 mmol) of trans-2-butenoic acidchloride and, after completion of the addition, the mixture was stirredfor 1 hr at 5° C. and for 16 h at ambient temperature. The solution wasdiluted with 50 ml of CH₂ Cl₂ and was successively washed with saturatedaqueous NaHCO₃, brine, 1% aqueous HCl and brine. The organic phase wasdried over Na₂ SO₄ and the solvent was evaporated under reduced pressureto provide 360 mg of a gum. This was dissolved in 50 ml of 95% ethanoland to it was added 5 ml of 2N HCl. After the solution had been storedat room temperature for 22 h, it was diluted with 100 ml of CH₂ Cl₂ andwas washed successively with H₂ O, saturated aqueous NaHCO₃ and brine.The organic phase was dried over Na₂ SO₄ and the solvent was evaporatedunder reduced pressure to provide 260 mg of gum. This waschromatographed on 20 g of silica gel using 1% methanol in CH₂ Cl₂ asthe solvent. The first product eluted was4β,15-bis-(trans-2'-butenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene(26 mg) followed by 22 mg of4β(trans-2'-butenoyloxy)-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene andthen 147 mg of the title compound which was crystallied from CHCl₃-SKELLYSOLVE B as a white solid of mp 83°-86°. IR(KBr): 3440, 2970,1725, 1190, 1110, 1085, 965 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.5H₂ O: C, 63.49; H, 7.57. Found: C, 63.54;H, 7.43.

EXAMPLE 54β,15-Bis-(trans-2'-butenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene

Repetition of the above experiment using 6 equivalents oftrans-2-butenoic acid chloride gave the title compound as a hygroscopicwhite foam. IR(KBr): 3420, 2970, 1720, 1310, 1260, 1185, 965 cm⁻¹. Inaddition, there was obtained4β(trans-2'-butenoyloxy)-3α,15-dihydroxy-12,13-epoxytrichothec-9-ene asa cream solid of m.p. 60°-62° C. IR(KBr): 3460, 2960, 1710, 1315, 1190,1105, 1080, 955 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.25H₂ O: C, 64.30; H, 7.53. Found: C,64.19; H, 8.06.

EXAMPLE 615-(2'-methylpropenoyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene

To a solution of 366 mg (1 mmol) of3α-O-(2'-tetrahydropyranyl)-4β,15-dihydroxy-12,13-epoxytrichothec-9-eneand 395 mg (5 mmol) of dry pyridine in 50 ml of methylene chloride(dried over 4 Å molecular sieves) was added with stirring 261 mg (2.5mmol) of freshly distilled 2-methylpropenoic acid chloride. The solutionwas stored at 22° C. for 17 h and was then treated with an additional261 mg (2.5 mmol) of the acid chloride. After a further 22 h at 22° C.,the solution was worked up as described in Example 4 and the residue waschromatographed on 20 g of silica gel. 2-Methylpropenoic acid anhydridewas eluted using 1% methanol in CH₂ Cl₂. The solvent was changed tomethanol to elute 230 mg of white foam which was hydrolyzed as describedabove (Example 4) to give 189 mg of a foam. This was chromatographed on20 g silica gel using 1% methanol in CH₂ Cl₂ as the solvent. Minorproducts were eluted and the solvent was changed to 20% methanol in CH₂Cl₂ to afford 116 mg (33%) of the title compound as a foam whichcrystallized from CH₂ Cl₂ -SKELLYSOLVE B as a pale pink solid of m.p.79°-81° C. IR(KBr): 3440, 2960, 1715, 1165, 1080, 955 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.5H₂ O: C, 63.49; H, 7.57. Found: C, 63.36;H, 7.40.

EXAMPLE 74β-(Chloroacetoxy)-15-(2'-methylpropenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-eneA.3α-O-(2'-Tetrahydropyranyl)-15-(2'-methylpropenoyloxy)-4β-hydroxy-12,13-epoxytrichothec-9-ene

A solution containing 3.66 g (0.01 mol) of3α-O-(2'-tetrahydropyranyl)-4β,15-dihydroxy-12,13-epoxytrichothec-9-ene,3.95 g (0.05 mol) of pyridine and 2.61 g (0.025 mol) of freshlydistilled 2-methylpropenoic acid chloride in 250 ml of dry methylenechloride was stirred for 16 h at 22° C. An additional 2.61 g (0.025 mol)of the acid chloride was added and stirring was continued for 6 h. Thesolution was diluted with CH₂ Cl₂ and was washed in succession withsaturated aqueous NaHCO₃, brine, 1% aqueous HCl and brine. The organicphase was dried over Na₂ SO₄ and the solvent evaporated under reducedpressure to give 5.36 g of an oil. This was chromatographed on 100 g ofsilica gel using 1% methanol in CH₂ Cl₂ as the solvent.2-Methylpropenoic acid anhydride was first eluted, followed by 615 mg ofa foam which was hydrolysed as before (Example 4) in 67.5 ml of 95%ethanol and 13.5 ml of 1N HCl. The usual work-up gave 590 mg of gum fromwhich, by chromatography, 198 mg of4β,15-bis-(2'-methylpropenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-enewas isolated as a hygroscopic foam IR(KBr): 3500, 2960, 1720, 1165,1080, 960 cm⁻¹ ; which was identified by its NMR spectrum. The nextfraction from this chromatography afforded4β-(2'-methylpropenoyloxy)-3α,15-dihydroxy-12,13-epoxytrichothec-9-eneas colorless crystals of m.p. 175°-176° C. IR(KBr): 3510, 3460, 2500,1690, 1330, 1300, 1170, 1080, 1060, 910, 900 cm⁻¹.

Anal. Calc'd for C₁₉ H₂₆ O₆.0.25H₂ O: C, 64.30; H, 7.53. Found: C,64.24; H, 7.14.

From the chromatographic separation of the tetrahydropyranyl ethers(above) there was next obtained 810 mg of a foam which wasre-chromatographed on fresh silica gel (20 g) using the same solventsystem to provide3α-O-(2'-tetrahydropyranyl)-15-(2'-methylpropenoyloxy)-4β-hydroxy-12,13-epoxytrichothec-9-eneas a foam.

B.4β-(Chloroacetoxy)-15-(2'-methylpropenoyloxy)-3α-hydroxy-12,13-epoxytrichothec-9-ene

To a stirred solution of 164 mg (0.38 mmol) of3α-O-(2'-tetrahydropyranyl)-15-(2'-methylpropenoyloxy)-4β-hydroxy-12,13-epoxytrichothec-9-enein 25 ml of dry CH₂ Cl₂ were added in succession 36 mg (0.46 mmol) ofpyridine and 78 mg (0.46 mmol) of chloroacetic anhydride. The solutionwas stored for 17 h at 22° C. The solution was worked up as before andhydrolysed as usual with 27 ml of 95% ethanol and 5.4 ml of 1N HCl.After work-up as before there was obtained a gum which was trituratedwith SKELLYSOLVE B to provide a hydroscopic solid of m.p. 58°-60° C.IR(KBr): 2960, 1755, 1715, 1320, 1295, 1165, 1085, 955 cm⁻¹.

Anal. Calc'd for C₂₁ H₂₇ ClO₇ : C, 59.08; H, 6.38. Found: C, 60.48; H,6.66.

EXAMPLE 815-(2'-Chloropropionyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene##STR19##

To a mixture of scirpentriol (1.12 g) in 2,6-lutidine (10 ml), there wasadded with cooling 1 ml of 2-chloropropionyl chloride. After 24 hours atroom temperature the solution was poured on ice, and the mixture wasextracted with 3×15 ml of ethyl acetate. The organic layer was washedwith water and dilute sodium bicarbonate. After evaporation of the driedsolvent, there remained 1.1 g of an oil. This was dissolved in 8 ml ofbenzene and placed on a silica gel column (Grace, 1.5×49 cm). The columnwas eluted with benzene containing increasing proportions of methanol.The title compound emerged with 2.5% methanol and appeared homogeneouson TLC plates (SiO₂) developed with ethyl acetate-toluene (3:1 v/v).Yield: 28.8 mg.

The NMR spectrum (CDCl₃, 100 MHz) of the product showed the followingpeaks:

    ______________________________________                                        ppm                                                                           ______________________________________                                        0.7 (s, 3H)      C-14                                                         1.7-2.0 (m, 10H) C-16, C-7, C-8, CH.sub.3 CHCl                                2.8-3.1 (dd, 2H) C-13                                                         3.7 (d, 1H)      C-2                                                          3.9-4.1 (dd, 2H) C-15                                                         4.3-4.5 (m, 3H)  C-3, C-4, C-11                                               4.4-4.6 ((g, 1H) CHClCH.sub.3                                                 5.5 (d, 1H)      C-10                                                         ______________________________________                                    

NMR showed that C-15 was acylated (C-15 protons appeared at 3.9-4.1ppm); protons at C-3 and C-4 appeared approximately at 4.5 ppmindicating that they were not acylated.

EXAMPLE 9 15-Benzoyloxy-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene##STR20##

3α,4β,15-Tribenzoyl-12,13-epoxytrichothec-9-ene (1.4 g) was hydrolyzedfor 4.5 hours in a solution of 230 ml of methanol, 19 ml of concentratedammonium hydroxide and 31 ml of water. The mixture was concentrated andextracted with benzene (3×50 ml). The volume was reduced to 25-30 mlwhich was placed on a silica gel column (Grace, 2×49 cm) and eluted withbenzene containing increments of methanol. The fractions were monitoredon TLC plates (SiO₂) with benzene-methanol (188:12 v/v) as developer.Spots were visualized with an alkaline permanganate spray. The fractionemerging at 2% methanol yielded 56 mg of the pure 4β,15-dibenzoate; at3% methanol 221 mg of the title compound was obtained.

NMR (CDCl₃, 100 MHz) showed the following peaks:

    ______________________________________                                        ppm                                                                           ______________________________________                                        0.8 (s, 3H)         C-14                                                      1.7 (s, 3H)         C-16                                                      2.0 (m, 4H)         C-7, C-8                                                  2.7-3.0 (dd, 2H)    C-13                                                      3.9-4.3 (m, 3H)     C-3, C-4, C-11                                            4.0-4.4 (m, 2H)     C-15                                                      5.5 (d, 1H)         C-10                                                      7.1-8 (m, 5H)       benzoyl                                                   ______________________________________                                    

C-3 and C-4 protons appeared at 3.9-4.3 ppm indicating that they werenot acylated.

The starting material,3α,4β,15-tribenzoyloxy-12,13-epoxytrichothec-9-ene, in this experimentwas prepared by acylating scirpentriol with benzoyl chloride accordingto the following procedure:

To scirpentriol (840 mg) dissolved in 6 ml of pyridine, there was added1.3 g of benzoyl chloride. After 24 hours at room temperature thesolution was added to ice. The usual work-up gave a solid which wascrystallized from ether-hexane to give 350 mg of3α,4β,15-tribenzoyloxy-12,13-epoxytrichothec-9,10-ene.

The NMR spectrum (CDCl₃, 100 MHz) of the product showed the followingpeaks:

    ______________________________________                                        ppm                                                                           ______________________________________                                        1.05 (s, 3H)          C-14                                                    1.75 (s, 3H)          C-16                                                    2.10 (m, 4H)          C-7, C-8                                                2.95-3.25 (dd, 2H)    C-13                                                    4.2 (d, 1H)           C-11                                                    4.5-4.8 (m, 2H)       C-15                                                    5.5-5.6 (d, 1H)       C-10                                                    5.7 (m, 1H)           C-3                                                     6.2 (d, 1H)           C-4                                                     7.3-8.2 (m, 15H)      (benzoyl)                                               ______________________________________                                    

The compound contains 3 benzoyl groups; protons at C-3, C-4 and C-15 areon carbons bearing acylated hydroxyl groups.

EXAMPLE 109,10β-Epoxy-15-(trans-2'-butenoyloxy)-3α,4β-dihydroxy-12,13-epoxytrichothec-9-ene##STR21##

A solution containing approximately equimolar amounts of the bis-esterof Example 5 and m-chloroperbenzoic acid in CH₂ Cl₂ was stirred at roomtemperature for 72 hours. The resulting solution was washed insuccession with 10% aqueous Na₂ SO₃, saturated aqueous NaHCO₃ and brine.The organic phase was dried over Na₂ SO₄ and the solvent was evaporatedunder reduced pressure to give the title product, m.p. 83°-85° C.

Anal. Calc'd. for C₁₉ H₂₆ O₇ 0.5H₂ O: C, 60.78; H, 7.25. Found: C,60.52; H, 7.25.

We claim:
 1. The compound having the formula ##STR22##